Project Summary Humans respond to tumor development by generating tumor-antigen-specific effector cytotoxic lymphocytes (CTLs) to eliminate tumor growth and development. However, tumor cells often mount a counterattack against CTLs by sensing activated T cell-produced IFN? as a danger signal. In an ?adaptive immune resistance mechanism,? tumor cells up-regulate PD-L1, suppressing PD-1+ CTLs in the tumor microenvironment. Therefore, tumor cells hijack the T cell-APC immune checkpoint system to evade host cancer immunosurveillance. Accordingly, anti-PD-1 antibody immune checkpoint inhibitor (ICI) immunotherapy has shown remarkable and durable efficacy in many types of human cancers. However, factors underlying a robust response to ICI therapy remain unclear, and human colorectal cancer, except for the small subset of microsatellite instable subtype cases (MSI), does not respond to ICI immunotherapy, suggesting that other immune checkpoints may suppresses CTL activation in the CRC tumor microenvironment. Interferon Regulatory Factor 8 (IRF8) was originally identified as a lineage-specific transcription factor for myeloid cell differentiation. Loss of IRF8 expression leads to accumulation of CD11b+Gr1+ immature myeloid cells in mice that phenotypically resemble myeloid-derived suppressor cells (MDSCs). IRF8 has recently emerged as a key regulator of T cell activation. In preliminary studies, we determined that IRF8 represses OPN expression in myeloid cells to regulate CD8+ T cell activation in a cell- extrinsic manner. Our central hypothesis is that the CD44-OPN axis is another immune checkpoint and that colon carcinoma cells overexpress OPN as a mechanism to suppress CTL activation in the tumor microenvironment. Our objectives are: 1) elucidate the molecular mechanism underlying IRF8 regulation of OPN expression in myeloid cells under physiological and pathological conditions; and 2) test the hypothesis the tumor cell-expressed OPN engages CTL-expressed CD44 to suppress CTL activation in the tumor microenvironment and to promote tumor progression. Successful completion of this project will not only establish CD44-OPN as a novel immune checkpoint but will also provide the mechanistic basis for the development of new therapies to overcome colorectal cancer resistance to immunotherapy.